Analysis and Antioxidant Activity of Extracts from Broccoli (Brassica

Jan 9, 2015 - ABSTRACT: Samples prepared from fresh broccoli (Brassica oleracea L.) sprouts by water distillation or freeze-drying were examined for ...
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Analysis and Antioxidant Activity of Extracts from Broccoli (Brassica oleracea L.) Sprouts Hae Won Jang,† Joon-Kwan Moon,§ and Takayuki Shibamoto* Department of Environmental Toxicology, University of California, Davis, California 95616, United States ABSTRACT: Samples prepared from fresh broccoli (Brassica oleracea L.) sprouts by water distillation or freeze-drying were examined for antioxidant activity using three assays. All samples exhibited dose-dependent antioxidant activity. The antioxidant activity ranged from 74.48 ± 0.46% (less volatile sample) to 93.2 ± 0.2% (dichloromethane extract sample) at the level of 500 μg/mL. Both dichloromethane extract samples from a water distillate of broccoli sprouts and freeze-dried broccoli sprouts showed potent antioxidant activity, which was comparable to that of BHT. Among the 43 compounds positively identified by gas chromatography−mass spectrometry, 5-methylthiopentylnitrile (31.64 μg/g) was found in the greatest concentration, followed by 4-methylthiobutylisothiocyanate (14.55 μg/g), 4-methylthiobutylnitrile (10.63 μg/g), 3-methylthiopropylisothiocyanate (3.00 μg/g), and 4-methylpentylisothiocyanate (2.48 μg/g). These isothiocyanates are known to possess antioxidant properties. Possible phenolic antioxidants found are 4-(1-methylpropyl)phenol (0.012 μg/g), 4-methylphenol (0.159 μg/g), and 2-methoxy4-vinylphenol (0.009 μg/g). The present study demonstrates that broccoli sprouts are a good source of natural antioxidants. KEYWORDS: broccoli sprouts, isothiocyanates, natural antioxidants, volatile compounds

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compounds was reported in the late 1990s,12 volatile antioxidants have begun to receive much more attention. Accordingly, many volatile antioxidants, such as monoterpenoids,13−16 have been found in foods and natural plants.17 Therefore, searching for volatile chemicals that may be present in foods and plants is an important part of identifying healthbeneficial foods and beverages. Moreover, natural medicinal plant essences containing volatile aroma chemicals have been used in medicinal aromatherapy since ancient times, suggesting that they offer beneficial health effects in addition to their pleasant odors. In the present study, volatile samples prepared from broccoli sprouts were examined for antioxidant activity and analyzed by gas chromatography (GC) and gas chromatography−mass spectrometry (GC-MS).

INTRODUCTION Broccoli (Brassica oleracea L.) is one of the most widely consumed vegetables in the United States. It contains various nutrients, including vitamins A, C, and K, as well as high levels of soluble fiber.1 Recently, broccoli sprouts have received a great deal of attention because their bioactive constituents are somewhat different from those of mature broccoli and are reportedly even more effective than broccoli in the prevention of diseases caused by reactive oxygen species.2,3 In addition, sprouts have a much shorter cultivation period than mature plants, suggesting that they can be harvested before being damaged by diseases. The production and consumption of broccoli sprouts are steadily increasing. Even though there are many papers focusing on the antioxidant activity of fully developed broccoli, only a little information on the antioxidant activities of broccoli sprouts is available today. Moreover, reports on the antioxidant activity of the volatile compounds from broccoli sprouts are not yet available. Previous studies on broccoli have demonstrated that it contains antioxidants, including carotenoids, αtocopherols, ascorbic acid, and phenolic compounds.4−6 These antioxidants are known to scavenge reactive oxygen species and protect cells from oxidative damage.7,8 Recently, natural plants have received a great deal of attention as a source of natural antioxidants because some synthetic antioxidants, such as butylated hydroxytoluene (BHT), exhibit toxic effects.9 If antioxidants can be found in broccoli sprouts, it would be possible to offer a speedier and more economical way to derive the health benefits already known to be associated with the consumption of broccoli. Over the past three decades, investigations into the antioxidants found in foods and natural plants have mainly focused on less volatile compounds or unextractable components, such as polyphenols10 and flavonoids.11 However, ever since the antioxidant activity of volatile heterocyclic © XXXX American Chemical Society

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MATERIALS AND METHODS

Chemicals and Materials. N-Methylhydrazine, 1-methylpyrazole, 2-methylpyrazine, 1,1-diphenyl-2-picrylhydrazyl (DPPH), sodium dodecyl sulfate, and ferrous chloride were purchased from Sigma Chemical Co. (Milwaukee, WI, USA). Cod liver oil (approximately 70% ω-3 fatty acid methyl esters), BHT, trizma hydrochloride, and trizma base were bought from Sigma Chemical Co. (St. Louis, MO, USA). Authentic chemicals were donated by Takata Koryo Co., Ltd. (Hyogo, Japan). All other chemicals and solvents were obtained from reliable commercial sources. 5-(Methylthio)pentylnitile, 3-(methylthio)propylisothiocyanate, and 4-(methylthio)butylisothiocyanate were synthesized according to a previously reported method.18 Broccoli sprout seeds were provided by Cosmo Salad Co., Ltd. (Dixon, CA, USA) as a gift and cultivated according to a previously reported method.19 Sprouts were harvested 7 days after germination. Received: October 16, 2014 Revised: December 3, 2014 Accepted: January 9, 2015

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Figure 1. Experimental scheme of sample preparations. each extract was removed by rotary flash evaporator, they were stored at −5 °C until used for the antioxidant tests. Analysis of Components in the Volatile Sample. Components in the volatile sample were isolated and identified by GC and GC-MS. Identification was conducted by comparing the Kovats index20 and MS fragmentation pattern of a GC component with those of an authentic chemical. The concentration of each compound in broccoli sprouts was calculated according to a previously reported method.15 Antioxidant Assays. Three assays were used to examine the antioxidant activity of the samples. Details of the assays used in the present study were described in a previous paper.21 A standard antioxidant, BHT, was used to validate the assays used. Malonaldehyde/Gas Chromatography (MA/GC) Assay. This assay is useful for examining the last stage of lipid oxidation.21 An aqueous assay solution (5 mL) containing 30 μL of cod liver oil, 0.25 mmol of trizma buffer (pH 7.4), 1 μmol of ferrous chloride, 2 μmol of hydrogen peroxide, 0.75 mmol of potassium chloride, and 1% surfactant sodium dodecyl sulfate was prepared. Various amounts of broccoli sprout samples were added to this assay solution and incubated for 17 h at 37 °C in a 20 mL test tube. After incubation, 50 μL of 4% BHT solution was added to terminate the oxidation of the samples. After MA formed from cod liver oil in the test tube, it was derivatized to 1-methylpyrazole with N-methylhydrazine (30 μL). The 1-methylpyrazole formed was eluted with 10 mL of ethyl acetate through an SPE cartridge (MEGA BE-C18, 1 g, 6 mL, Varian, USA). After 20 μL of GC internal standard was added, 2-methylpyrazine was added to the eluate, and the volume of the eluate was adjusted to exactly 10 mL with ethyl acetate. The eluate solution was analyzed for 1-methylpyrazole by GC. All experiments were carried out in triplicate. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Assay. The DPPH solution (1.52 × 10−4 M) was prepared in ethanol. DPPH solution (4.5 mL) and 0.5 mL of test sample were mixed using a vortex and stored in the dark for 20 min. After the reaction ran to completion, the absorption

Sample Preparations for Analysis and Antioxidant Assays. The entire scheme of the experimental procedures in the present study is shown in Figure 1. Water Distillation Samples. Fresh broccoli sprouts (500 g) were placed in a 3 L round-bottom flask with 1 L of deionized water. The aqueous solution was water-distilled at 50 °C for 5 h under reduced pressure (95 mmHg). The distillate (900 mL) was extracted with 250 mL dichloromethane using a liquid−liquid continuous extractor for 6 h. After the extract was dried over anhydrous sodium sulfate, the solvent was removed by a rotary flash evaporator and then placed under a purified nitrogen stream to yield brown viscous material (31.9 mg). This material, labeled the volatile sample, was set aside to be used further for the antioxidant tests and GC-MS analysis. The residual broccoli sprouts in the distillation flask were mixed with 200 mL of methanol by stirring for 2 h and then filtered. The solvent of the filtrate was removed by rotary flash evaporator, and residual materials were freeze-dried (as the less-volatile sample) and stored at 4 °C until used in the antioxidant tests. Fractionation of the Volatile Sample. The volatile sample was placed in a glass column (15 cm × 1 cm i.d.) packed with 5 g of silica gel (100−200 mesh, Avantor Performance Materials, Inc., Philipsburg, NJ, USA) and eluted sequentially with the following 100 mL pentane/ ethyl acetate solvent fractions: 100/0 (fraction I), 95/5 (fraction II), 80/20 (fraction III), 50/50 (fraction IV), 20/80 (fraction V), 0/100 (fraction VI); finally, elution with 200 mL of acetone yielded fraction VII. The solvent of each fraction was removed by a rotary flash evaporator and then by a purified nitrogen stream to 1 mL in volume. The seven fractions prepared were stored at 4 °C until used for the antioxidant tests and GC-MS analysis. Freeze-Dried Sample. Fresh broccoli sprouts were freeze-dried at −48 °C for 48 h (FreezOne, Labconco, MO, USA). The freeze-dried broccoli sprouts (40 g) were extracted sequentially with 1 L each of hexane, dichloromethane, and acetone using a Soxhlet extractor. The extracts were dried over anhydrous sodium sulfate. After the solvent of B

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Journal of Agricultural and Food Chemistry activity was measured at 517 nm using a UV−visible spectrophotometer. A solution without the testing sample was used as the control. All experiments were conducted in triplicate. Aldehyde/Carboxylic Acid Assay. This method is useful to test the long-term antioxidant activity, which is important for determining the shelf life of a product.21 Various amounts of the volatile sample (refer to Figure 1) were added to a 2 mL dichloromethane solution of hexanal (3 mg/mL) containing 0.2 mg/mL undecane as a GC internal standard. Standard antioxidant BHT was used to validate the method. Oxidation of the sample solution was initiated by heating at 60 °C for 10 min in a sealed vial and then stored at room temperature. The oxidation of hexanal to hexanoic acid was monitored at 5 day intervals by measuring the amount of hexanal using GC. All experiments were performed in triplicate. Instruments. An Agilent model 6890 GC equipped with a 60 m × 0.32 mm i.d. (df = 0.25 μm) DB-WAX bonded-phase fused-silica capillary column (Agilent, Folsom, CA, USA) and an FID were used for obtaining a Kovats index of components in the volatile sample and its fractions (refer to Figure 1). The linear velocity of the helium carrier gas was 28 cm/s at a split ratio of 10:1. The injector and detector temperatures were 260 and 300 °C, respectively. The oven temperature was programmed to increase from 50 to 210 °C at 2 °C/ min and held for 30 min. An Agilent model 6890 GC interfaced to a 5973 mass selective detector (GC-MS) was used for mass spectral identification of the GC components at an MS ionization voltage of 70 eV. GC column conditions were exactly the same as the ones used for GC-FID. The quantitative analysis of 1-methylpyrazole for the MA/GC assay was performed using an Agilent model 6890 GC equipped with a 30 m × 0.32 mm i.d. (df = 0.25 μm) DB-WAX bonded-phase fused silica capillary column (Agilent) and an NPD. The injector and detector temperatures were 250 °C. The oven temperature was programmed to increase from 60 to 160 °C at 4 °C/min and held for 2 min. The linear velocity of the helium carrier gas was 28 cm/s at a split ratio of 20:1. The quantitative analysis of hexanal for the aldehyde/carboxylic acid assay was conducted by using an Agilent model 6890 GC equipped with a 30 m × 0.25 mm i.d. (df = 0.25 μm) DB-1 bonded-phase fusedsilica capillary column (Agilent) and a FID. The linear velocity of the helium carrier gas was 30 cm/s at a split ratio of 20:1. The injector and detector temperatures were 300 and 280 °C, respectively. The oven temperature was programmed to rise from 40 to 180 °C at 5 °C/min and then held for 10 min. Statistical Analyses. The data obtained from the present study were treated with an ANOVA analysis based on the SAS system, and the level of significance was determined using Duncan’s multiple-range test at α = 0.05.

Figure 2. Results of MA/GC assay on the samples prepared from broccoli sprouts by freeze-drying and water distillation.

that all samples contain strong antioxidants. The volatile sample showed 82.1 ± 2.4% at the level of 500 μg/mL. Among the samples tested in this experiment, the sample prepared by Soxhlet extraction with dichloromethane solvent from the freeze-dried sample (refer to Figure 1) exhibited the most potent antioxidant activity. Its antioxidant activities (87.9 ± 1.9% at 100 μg/mL and 93.2 ± 0.6% at 500 μg/mL) were comparable to that of BHT. Figure 3 shows the results of the DPPH assay. Values are the mean ± SD (n = 3). The results are consistent with those of the

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Figure 3. Results of DPPH assay on the samples prepared from broccoli sprouts by freeze-drying and water distillation.

RESULTS AND DISCUSSION Antioxidant Activities of Samples Prepared by Freeze-Drying and Water Distillation. Whereas spectrophotometric methods, such as DPPH assay, cause some difficulties because the absorption of color materials overlaps with that of the target chemical, the MA/GC assay can be applied to samples with dark colors because it measures MA specifically by GC. Therefore, the MA/GC method was used for the preliminary examination of the samples prepared from broccoli sprouts (refer to Figure 1). Figure 2 shows the antioxidant activity of the samples examined by the MA/GC assay. The values are the mean ± SD (n = 3). BHT showed antioxidant activities of 95.3 ± 0.2% at the level of 500 μg/mL, 92.2 ± 0.4% at the level of 100 μg/mL, and 87.2 ± 3.1% at the level of 20 μg/mL, confirming that the method used was valid. All samples exhibited dose-dependent antioxidant activity. The antioxidant activity ranged from 74.48 ± 0.46% (less volatile sample) to 93.2 ± 0.2% (dichloromethane extract sample) at the level of 500 μg/mL, indicating

MA/GC assay. The volatile sample was not tested in this experiment because it was tested by the aldehyde/carboxylic acid assay. BHT exhibited clear antioxidant activities with dose response effects: 85.0 ± 1.0% at the level of 500 μg/mL, 47.3 ± 3.9% at the level of 100 μg/mL, and 10.8 ± 0.2% at the level of 20 μg/mL, confirming that this assay is valid. Less volatile samples and acetone samples showed slight dose-dependent antioxidant activities: 30.42 ± 0.17 and 31.7 ± 0.8% at the level of 500 mg/mL, respectively. Dichloromethane samples showed the most potent antioxidant activity among the samples tested in this experiment, which was the same as the results obtained by the MA/GC assay. The antioxidant activity of a dichloromethane sample (83.3 ± 1.7%) was comparable to that of BHT (85.0 ± 1.0%) at the level of 500 μg/mL. Also, dichloromethane and hexane samples exhibited moderate antioxidant activities at the level of 100 μg/mL, which were 39.1 ± 1.2 and C

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Figure 4. Results of aldehyde/carboxylic acid assay on the volatile sample.

51.8 ± 0.6%, respectively. The less volatile and acetone samples showed only slight antioxidant activities (30.42 ± 0.17 and 31.7 ± 0.8% at the level of 500 mg/mL). These results from two antioxidant assays suggest that potent antioxidants are present in the dichloromethane and hexane samples and that the amounts of water-soluble antioxidants are less than those of organic solvent extractable (volatile) antioxidants in broccoli sprouts. Figure 4 shows the antioxidant activity of the volatile sample examined by the aldehyde/carboxylic acid assay over 30 days. This assay is useful in discovering the long-term antioxidant activities of a chemical or a group of chemicals.21 The values are the mean ± standard deviation (SD) (n = 3). SD ranged from 0.88 (500 μg/mL sample at 10 days) to 34.64 (control sample at 10 days). SDs lower than 1.0 do not appear in the figure. BHT showed >99% antioxidant activity throughout the experiment. The volatile sample exhibited dose-dependent antioxidant activity. The control sample was completely oxidized after 30 days. By the 30th day, the antioxidant activity of the samples was 8% by 1 μg/mL, 29% by 5 μg/mL, 70% by 10 μg/mL, 98% by 50 μg/mL, and 99% by 100 and 500 μg/ mL. The 50 and 100 μg/mL samples exhibited antioxidant activity comparable to that of BHT. The results suggest that broccoli sprouts contain antioxidants, the activity of which is maintained for prolonged periods. Figure 5 shows the antioxidant activity of the fractions obtained from the volatile sample tested by the aldehyde/ carboxylic acid assay after 30 days. Values are the mean ± SD (n = 3). BHT exhibited 99.84 ± 2.49% at the level of 10 μg/ mL. Fractions I, II, and III exhibited potent dose-dependent antioxidant activity. In particular, fraction II showed activity (95.56 ± 0.22%) comparable with that of BHT at the level of 10 μg/mL. On the other hand, fractions IV, V, VI, and VII exhibited only slight antioxidant activities at the levels tested in this experiment. These results suggest the presence of volatile nonpolar antioxidants in broccoli sprouts. According to the results obtained in the present study, samples prepared with dichloromethane seem to contain potent antioxidants. Therefore, the volatile sample was analyzed to determine the principal antioxidants in the sprouts. Compounds Identified in the Volatile Sample. Table 1 shows the compounds identified in the volatile sample from

Figure 5. Results of aldehyde/carboxylic acid assay after 30 days on fractions obtained from the volatile sample.

broccoli sprouts, along with their calculated concentrations (μg/g of broccoli sprouts) and Kovats index. Among the over 88 GC peaks observed in the gas chromatogram, 43 compounds were positively identified. The major compounds are 4-methylpentylisothiocyanate (2.48 μg/g), 4-(methylthio)butylnitrile (10.63 μg/g), 5-(methylthio)pentylnitrile (31.64 μg/g), 3-(methylthio)propylisothiocyanate (3.00 μg/g), and 4(methylthio)butylisothiocyanate (14.55 μg/g). One recent study reported that these compounds possess strong antiHelicobacter activity.18 Dimethyl disulfide, 1-penten-3-ol, 2,4,5-trimethylthiazole, 3(methylthio)propanal, allylisothiocyanate, 2-ethyl-1-hexanol, phenylethyl alcohol, 2-methoxy-4-vinylphenol, and 2-isothiocyanatoethylbenzene have also been reported as volatile components in various vegetables and fruits, including soybeans, mung beans, onions/sprouts, horseradish, cumin, kale sprouts, and persimmon, and some organic solvent extracts of these plants showed certain antioxidant activities.22−24 When a headspace of raw and cooked broccoli was analyzed, dimethyl sulfide, dimethyl trisulfide, hexanal, and nonanal, which contribute to the characteristic aroma of broccoli, were D

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not in mature broccoli. The functional group −NCS in isothiocyanate compounds has been known to possess antioxidant activity, which induces phase II enzymes associated with detoxification of chemical carcinogens.28 It is well-known that phenolic compounds found in plants possess antioxidant activity.29 Possible phenolic antioxidants found in the present study are 4-(1-methylpropyl)phenol, 4methylphenol, and 2-methoxy-4-vinylphenol. A potent antioxidant activity of 2-methoxy-4-vinylphenol isolated and identified from a persimmon peel was previously reported.30 Moreover, 2-methoxy-4-vinylphenol found in brewed coffee possessed potent antioxidant activity (>90%) at the level of 200 μg/mL tested by MA/GC assay.31 Fraction I (refer to Figure 1) contained isobutylisothiocyanate (0.03 μg/g), allylisothiocyanate (0.02 μg/g), and 2phenylethylisothiocyanate (0.73 μg/g). A previous study has demonstrated that allylisothiocyanate was found as a volatile compound in Emeraude cauliflower and possessed strong antioxidant activity.32 Fraction II contained 4-methylpentylisothiocyanate (0.31 μg/g), 4-(methylthio)butylnitrile (3.70 μg/ g), 5-(methylthio)pentylnitrile (7.15 μg/g), 3-(methylthio)propylisothiocyanate (3.20 μg/g), and 4-(methylthio)butylisothiocyanate (9.98 μg/g). 4-(Methylthio)butylisothiocyanate has direct antioxidant activity in addition to inducing phase II enzymes. It also oxidizes to sulforaphane, which has anticancer, anti-Helicobacter, and antioxidant activities.18,33 4-Methylpentylisothiocyanate found in Raphanus sativus root induced apoptotic cell death in human cancer cell lines.34 4-(Methylthio)butylnitrile, 5-(methylthio)pentylnitrile, and 3-(methylthio)propylisothiocyanate were also reported in Emeraude and Magnifico cauliflower as major volatile compounds.32 Fraction III also contained high levels of 4(methylthio)butylnitrile (8.26 μg/g) and 5-(methylthio)pentylnitrile (24.98 μg/g), which may have contributed to the antioxidant activity of this fraction. These nitrogen/sulfurcontaining compounds comprised >90% of the volatile sample and may significantly contribute to the antioxidant activity of broccoli sprouts. The less volatile sample (refer to Figure 1) also showed relatively moderate antioxidant activity with dose response effects as shown in Figures 2 and 3, suggesting the presence of water-soluble antioxidants. Therefore, the total antioxidant activity of the samples prepared from broccoli sprouts may be comparable to those of known antioxidants such as αtocopherol and BHT. The present study demonstrated that broccoli sprouts have a strong potential to be a valuable source of antioxidants. Moreover, broccoli sprouts have a short harvest time (2 weeks) and are relatively easy to produce. Therefore, broccoli sprouts could be a good source for natural antioxidants.

Table 1. Compounds Identified in a Volatile Sample Kovats indexa concentration (μg/g) alkyl-hydrocarbons, -carbonyls, -alcohols, 1-penten-3-ol (E)-2-hexene 3-hydroxy-2-butanone 2-penten-1-ol 1-hexanol nonanal 2-butoxyethanol 2-ethyl-1-hexanol (E,E)-2,4-heptendienal 3-hydroxy-2,4,4-trimethylpentyl 2-methylpropanoate aromatic compounds 1,2-dimethoxybenzene phenylethyl alcohol 4-(1-methylpropyl)phenol 4-methylphenol 2-methoxy-4-vinylphenol thiocyanate and isothiocyanates methyl thiocyanate isobutyl isothiocyanate allylisothiocyanate 4-methylsulfinylbutylisothiocyanate 1-isothiocyanate-3-methylbutane 4-methylpentylisothiocyanate 3-(methylthio)propylisothiocyanate 4-methylphenyl isothiocyanate 4-(methylthio)butylisothiocyanate 2-phenylethylisothioxyanate nitriles 4-methylpentenenitrile 3-(methylthio)propanenitrile 4-(methylthio)butylnitrile benzyl nitrile benzenepropanenitrile 5-(methylthio)pentylnitrile sulfides dimethyl sulfide 2,4-dithiapentane dimethyl trisulfide 3-(methylthio)propanal 3-(methylthio)propanol 4-(methylthio)-1-butanal heterocyclic compounds tetrahydrothiophene pyridine 1,4,5-trimethylthiazole dihydro-2(3)-thiophenone 1,2,4-trithiolane a

and esters 1163 1220 1281 1314 1350 1390 1402 1486 1494 1868

0.056 0.011 0.013 0.163 0.016 0.008 0.129 0.029 0.022 0.115

1708 1899 2006 2054 2166

0.013 0.024 0.012 0.159 0.009

1264 1335 1352 1422 1431 1549 1981 2090 2132 2215

0.119 0.003 0.025 0.003 0.007 2.481 3.003 0.028 14.547 0.798

1244 1703 1785 1915 2030 1934

0.034 0.028 10.627 0.062 0.482 31.639

1082

0.004

1364 1450 1713 1831

0.018 0.080 0.011 0.181

1152 1186 1358 1638 1728

0.084 0.031 0.020 0.020 0.034

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On DB-WAX.

AUTHOR INFORMATION

Corresponding Author

*(T.S.) Phone: (530) 752-4523. Fax: (530) 752-3394. E-mail: [email protected].

identified.25 4-(Methylthio)butylisothiocyanate, 2-phenylethylisothiocyanate, 5-(methylthio)pentylnitrile, and 1,2-dimethoxybenzene were also reported in raw broccoli in a previous study.26 In broccoli stored under low-oxygen conditions, 3hydroxybutan-2-one, 2-pentenol, hexanal, hexanol, (E,E)-2,4heptadienal, and phenylpropanenitrile were identified.27 In this study, 4-methylpentylisothiocyanate, 3-(methylthio)propylisothiocyanate, and 4-(methylthio)butylnitrile were found as the major compounds found in broccoli sprouts but

Present Addresses †

(H.W.J.) Korea Food Research Institute, 1201-62 Anyangpangyoro, Bundanggu, Seongnamsi, Gyeonggido 463-746, Republic of Korea. § (J.-K.M.) Department of Plant Life and Environmental Sciences, Hankyong National University, Ansung, Republic of Korea. E

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(16) Lee, K. G.; Shibamoto, T. Determination of antioxidant properties of aroma extracts from various beans. J. Agric. Food Chem. 2000, 48, 4817−4820. (17) Shiratsuchi, H.; Chang, S.; Wei, A.; El-Ghorab, A. H.; Shibamoto, T. Biological activities of low-molecular weight compounds found in foods and plants. J. Food Drug Anal. 2012, 20 (Suppl. 1), 359−365. (18) Moon, J. K.; Kim, J. R.; Ahn, Y. J.; Shibamoto, T. Analysis and anti-Helicobacter activity of sulforaphane and related compounds present in broccoli (Barassica oleracea L.) sprouts. J. Agric. Food Chem. 2010, 58, 6672−6677. (19) Kamiyama, M.; Shibamoto, T. Increase of bioactive sulforaphane and its related compounds with sulfur compounds in broccoli (Brassica oleracea var. italica) sprouts during cultivation. Int. J. Agric. Innovations Res. 2013, 2, 299−306. (20) Kovàts, E. Gas chromatographic characterization of organic substances in the retention index system. Adv. Chromatogr. 1965, 1, 229−247. (21) Moon, J.-K.; Shibamoto, T. Antioxidant assays for plant and food components. J. Agric. Food Chem. 2009, 57, 1655−1666. (22) Bettaieb, I.; Bourgou, S.; Wannes, W. A.; Hamrouni, I.; Limam, F.; Marzouk, B. Essential oils, phenolic, and antioxidant activities of different parts of cumin (Cuminum cyminum L.). J. Agric. Food Chem. 2010, 58, 10410−10418. (23) Lee, K. G.; Shibamoto, T. Antioxidant properties of aroma compounds isolated from soybean and mung bean. J. Agric. Food Chem. 2000, 48, 4290−4293. (24) Takahashi, M.; Shibamoto, T. Chemical compositions and antioxidant/anti-inflammatory activities of steam distillate from freezedried onion (Allium cepa L.) sprout. J. Agric. Food Chem. 2008, 56, 10462−10467. (25) Jacobsson, A.; Nielsen, T.; Sjöholm, I. Influence of temperature, modified atmosphere packaging, and heat treatment on aroma compounds in broccoli. J. Agric. Food Chem. 2004, 52, 1607−1614. (26) Buttery, R. G.; Guadagni, D. G.; Ling, L. C.; Seifert, R. M.; Lipton, W. Additional volatile components of cabbage, broccoli, and cauliflower. J. Agric. Food Chem. 1976, 24, 829−832. (27) Krunbein, A.; Klaring, H. P.; Schonhof, I.; Schreiner, M. Atmospheric carbon dioxide changes photochemical activity, soluble sugars and volatile levels in broccoli (Brassica oleracea var. italica). J. Agric. Food Chem. 2010, 58, 3747−3752. (28) de Figueiredo, S. M.; Fiho, S. A.; Nogueira-Machado, J. A.; Caligiorne, R. B. The anti-oxidant properties of isothiocyanates: a review. Recent Pat. Endocr. Metab. Immune Drug Discovery 2013, 7, 213−225. (29) Rubió, L.; Motilva, M. J.; Romero, M. P. Recent advances in biologically active compounds in herbs and spices: a review of the most effective antioxidant and anti-inflammatory active principles. Crit. Rev. Food Sci. Nutr. 2013, 53, 943−953. (30) Fukai, S.; Tanimoto, S.; Maeda, A.; Fukuda, H.; Okada, Y.; Nomura, M. Pharmacological activity of compounds extracted from persimmon peel (Diospyros kaki THUNB.). J. Oleo Sci. 2009, 58, 213− 219. (31) Fujioka, K.; Shibamoto, T. Quantitation of volatiles and nonvolatile acids in an extract from coffee beverages: correlation with antioxidant activity. J. Agric. Food Chem. 2006, 54, 6054−6058. (32) Podsedek, A. Natural antioxidants and antioxidant capacity of Brassica vegetables: a review. LWT 2007, 40, 1−11. (33) Barillari, J.; Canistro, D.; Paolini, M.; Ferroni, F.; Pedulli, G. F.; Iori, R.; Valgimigli, L. Direct antioxidant activity of purified glucoerucin, the dietary secondary metabolite contained in rocket (Eruca sativa Mill.) seeds and sprouts. J. Agric. Food Chem. 2005, 53, 2475−2482. (34) Beevi, S. S.; Mangamoori, L. N.; Subathra, M.; Edula, J. R. Hexane extract of Raphanus sativus L. roots Inhibits cell proliferation and induces apoptosis in human cancer cells by modulating genes related to apoptotic pathway. Plant Foods Hum. Nutr. 2010, 65, 200− 209.

This work was supported in part by a research grant from Hankyong National University in the year of 2012. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED BHT, butylated hydroxytoluene; DPPH, 1,1-diphenyl-2-picrylhydrazyl; FID, flame ionization detector; GC, gas chromatography; GC−MS, gas chromatography−mass spectrometry; MA/GC assay, malonaldehyde/gas chromatography assay; NPD, nitrogen−phosphorus detector; SD, standard deviation; SPE, solid phase extraction

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REFERENCES

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DOI: 10.1021/jf504929m J. Agric. Food Chem. XXXX, XXX, XXX−XXX